Muscle Derived Cells for the Treatment of Gastro-Esophageal Pathologies and Methods of Making and Using the Same

ABSTRACT

The present invention provides muscle-derived progenitor cells that show long-term survival following transplantation into body tissues and which can augment soft tissue following introduction (e.g. via injection, transplantation, or implantation) into a site of soft tissue. Also provided are methods of isolating muscle-derived progenitor cells, and methods of genetically modifying the cells for gene transfer therapy. The invention further provides methods of using compositions comprising muscle-derived progenitor cells for the augmentation and bulking of mammalian, including human, soft tissues in the treatment of various cosmetic or functional conditions, including malformation, injury, weakness, disease, or dysfunction. In particular, the present invention provides treatments and amelioration of symptoms for gastro-esophageal pathologies like gastro-esophageal reflux.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 15/801,737, filed Nov. 2, 2017, which applicationis a continuation application of U.S. patent application Ser. No.15/067,351, filed Mar. 11, 2016, which is a continuation application ofU.S. patent application Ser. No. 13/766,894, filed Feb. 14, 2013, whichis a continuation application of U.S. patent application Ser. No.13/550,381, filed Jul. 16, 2012, which is a continuation application ofU.S. patent application Ser. No. 12/547,149, filed on Aug. 25, 2009,which claims benefit of priority from U.S. Provisional PatentApplication No. 61/091,569, filed on Aug. 25, 2008, and which is acontinuation-in-part of U.S. patent application Ser. No. 11/959,054 (nowU.S. Pat. No. 9,121,009) filed on Dec. 18, 2007, which claims thebenefit of priority from U.S. Provisional Application No. 60/870,516,filed on Dec. 18, 2006, contents of each of which are incorporatedherein by reference in their entireties.

GOVERNMENT INTERESTS

This invention was made with Government support under Grant No.DK0055387 awarded by the National Institutes of Health. The Governmenthas certain rights to this invention.

FIELD OF THE INVENTION

The present invention relates to muscle-derived progenitor cells (MDC)and compositions of MDCs and their use in the augmentation of bodytissues, particularly soft tissue like gastric and esophageal tissue. Inparticular, the present invention relates to muscle-derived progenitorcells that show long-term survival following introduction into softtissues, methods of isolating MDCs, and methods of using MDC-containingcompositions for the augmentation of human or animal soft tissues,including gastric and esophageal tissue. The invention also relates tonovel uses of muscle-derived progenitor cells for the treatment offunctional conditions, such as gastro-esophageal reflux disease.

BACKGROUND OF THE INVENTION

Augmentation of soft tissue using synthetic materials such as siliconeor polytetrafluoroethylene (PTFE) is well known in the art. U.S. Pat.No. 5,876,447 to Arnett discloses the use of silicone implants forfacial plastic surgery. However, such synthetic materials are foreign tothe host tissue, and cause an immunological response resulting in theencapsulation of the implant and scarring of the surrounding tissues.Thus, the implant may produce additional functional or aestheticproblems.

Soft tissue augmentation using biopolymers such as collagen orhyaluronic acid has also been described. For example, U.S. Pat. No.4,424,208 to Wallace et al. discloses methods of augmenting soft tissueutilizing collagen implant material. In addition, U.S. Pat. No.4,965,353 to della Valle et al. discloses esters of hyaluronic acid thatcan be used in cosmetic surgery. However, these biopolymers are alsoforeign to the host tissue, and cause an immunological responseresulting in the reabsorption of the injected material. Biopolymers aretherefore unable to provide long-term tissue augmentation. Overall, theuse of biopolymers or synthetic materials has been wholly unsatisfactoryfor the purpose of augmenting soft tissue.

Soft tissue augmentation using cell-based compositions has also beendeveloped. U.S. Pat. No. 5,858,390 to Boss, Jr. discloses the use ofautologous dermal fibroblasts for the treatment of cosmetic andaesthetic skin defects. Although this treatment avoids the problemsinherent in the implantation or injection of synthetic materials orbiopolymers, it results in other complications. Because fibroblastsproduce collagen, the cells can cause the stiffening and distortion ofthe tissues surrounding the implant site.

The use of autologous fat cells as an injectable bulking agent has alsobeen described (For review, see K. Mak et al., 1994, Otolaryngol. Clin.North. Am. 27:211 22; American Society of Plastic and ReconstructiveSurgery: Report on autologous fat transplantation by the ad hoccommittee on new procedures, 1987, Chicago: American Society of Plasticand Reconstructive Surgery; A. Chaichir et al., 1989, Plast. Reconstr.Surg. 84: 921 935; R. A. Ersek, 1991, Plast. Reconstr. Surg. 87:219 228;H. W. Horl et al., 1991, Ann. Plast. Surg. 26:248 258; A. Nguyen et al.,1990, Plast. Reconstr. Surg. 85:378 389; J. Sartynski et al., 1990,Otolaryngol. Head Neck Surg. 102:314 321. However, the fat graftingprocedure provides only temporary augmentation, as injected fat isreabsorbed into the host. In addition, fat grafting can result in noduleformation and tissue asymmetry.

Endoscopic delivery of bulking material has been tried for patientsuffering from gastro-esophageal reflux disease. However, as the recentrecall of ENTERYX® by the FDA suggests, there is a need for a safertreatment of this disease.

Myoblasts, the precursors of muscle fibers, are mononucleated musclecells that fuse to form post-mitotic multinucleated myotubes, which canprovide long-term expression and delivery of bioactive proteins (T. A.Partridge and K. E. Davies, 1995, Brit. Med. Bulletin 51:123 137; J.Dhawan et al., 1992, Science 254: 1509 12; A. D. Grinnell, 1994, MyologyEd 2, A. G. Engel and C. F. Armstrong, McGraw-Hill, Inc., 303 304; S.Jiao and J. A. Wolff, 1992, Brain Research 575:143 7; H. Vandenburgh,1996, Human Gene Therapy 7:2195 2200).

Cultured myoblasts contain a subpopulation of cells that show some ofthe self-renewal properties of stem cells (A. Baroffio et al., 1996,Differentiation 60:47 57). Such cells fail to fuse to form myotubes, anddo not divide unless cultured separately (A. Baroffio et al., supra).Studies of myoblast transplantation (see below) have shown that themajority of transplanted cells quickly die, while a minority survive andmediate new muscle formation (J. R. Beuchamp et al., 1999, J. Cell Biol.144:1113 1122). This minority of cells shows distinctive behavior,including slow growth in tissue culture and rapid growth followingtransplantation, suggesting that these cells may represent myoblast stemcells (J. R. Beuchamp et al., supra).

Myoblasts have been used as vehicles for gene therapy in the treatmentof various muscle- and non-muscle-related disorders. For example,transplantation of genetically modified or unmodified myoblasts has beenused for the treatment of Duchenne muscular dystrophy (E. Gussoni etal., 1992, Nature, 356:435 8; J. Huard et al., 1992, Muscle & Nerve,15:550 60; G. Karpati et al., 1993, Ann. Neurol., 34:8 17; J. P.Tremblay et al., 1993, Cell Transplantation, 2:99 112; P. A. Moisset etal., 1998, Biochem. Biophys. Res. Commun. 247:94 9; P. A. Moisset etal., 1998, Gene Ther. 5:1340 46). In addition, myoblasts have beengenetically engineered- to produce proinsulin for the treatment of Type1 diabetes (L. Gros et al., 1999, Hum. Gen. Ther. 10:1207 17); Factor IXfor the treatment of hemophilia B (M. Roman et al., 1992, Somat. Cell.Mol. Genet. 18:247 58; S. N. Yao et al., 1994, Gen. Ther. 1:99 107; J.M. Wang et al., 1997, Blood 90:1075 82; G. Hortelano et al., 1999, Hum.Gene Ther. 10:1281 8); adenosine deaminase for the treatment ofadenosine deaminase deficiency syndrome (C. M. Lynch et al., 1992, Proc.Natl. Acad. Sci. USA, 89:1138 42); erythropoietin for the treatment ofchronic anemia (E. Regulier et al., 1998, Gene Ther. 5:1014 22; B. Dalleet al., 1999, Gene Ther. 6:157 61), and human growth hormone for thetreatment of growth retardation (K. Anwer et al., 1998, Hum. Gen. Ther.9:659 70).

Myoblasts have also been used to treat muscle tissue damage or disease,as disclosed in U.S. Pat. No. 5,130,141 to Law et al., U.S. Pat. No.5,538,722 to Blau et al., and application U.S. Ser. No. 09/302,896 filedApr. 30, 1999 by Chancellor et al. In addition, myoblast transplantationhas been employed for the repair of myocardial dysfunction (C. E. Murryet al., 1996, J. Clin. Invest. 98:2512 23; B. Z. Atkins et al., 1999,Ann. Thorac. Surg. 67:124 129; B. Z. Atkins et al., 1999, J. Heart LungTransplant. 18:1173 80).

In spite of the above, in most cases, primary myoblast-derivedtreatments have been associated with low survival rates of the cellsfollowing transplantation due to migration and/or phagocytosis. Tocircumvent this problem, U.S. Pat. No. 5,667,778 to Atala discloses theuse of myoblasts suspended in a liquid polymer, such as alginate. Thepolymer solution acts as a matrix to prevent the myoblasts frommigrating and/or undergoing phagocytosis after injection. However, thepolymer solution presents the same problems as the biopolymers discussedabove. Furthermore, the Atala patent is limited to uses of myoblasts inonly muscle tissue, but no other tissue.

Thus, there is a need for other, different soft tissue augmentationmaterials that are long-lasting, compatible with a wide range of hosttissues, and which cause minimal inflammation, scarring, and/orstiffening of the tissues surrounding the implant site. Accordingly, themuscle-derived progenitor cell-containing compositions of the presentinvention are provided as improved and novel materials for augmentingsoft tissues. Further provided are methods of producing muscle-derivedprogenitor cell compositions that show long-term survival followingtransplantation, and methods of utilizing MDCs and compositionscontaining MDCs to treat various aesthetic and/or functional defects,including, for example, dermatological conditions or injury, and muscleweakness, injury, disease, or dysfunction.

It is notable that prior attempts to use myoblasts for non-muscle softtissue augmentation were unsuccessful (U.S. Pat. No. 5,667,778 toAtala). Therefore, the findings disclosed herein are unexpected, as theyshow that the muscle-derived progenitor cells according to the presentinvention can be successfully transplanted into non-muscle and musclesoft tissue, including epithelial tissue, and exhibit long-termsurvival. As a result, MDCs and compositions comprising MDCs can be usedas a general augmentation material for muscle or non-muscle soft tissueaugmentation, as well as for bone production. Moreover, since themuscle-derived progenitor cells and compositions of the presentinvention can be derived from autologous sources, they carry a reducedrisk of immunological complications in the host, including thereabsorption of augmentation materials, and the inflammation and/orscarring of the tissues surrounding the implant site.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide novel muscle-derivedprogenitor cells (MDCs) and MDC compositions exhibiting long-termsurvival following transplantation. The MDCs of this invention andcompositions containing the MDCs comprise early progenitor muscle cells,i.e., muscle-derived stem cells, that express progenitor cell markers,such as desmin, M-cadherin, MyoD, myogenin, CD34, and Bcl-2. Inaddition, these early progenitor muscle cells express the Flk-1, Sca-1,MNF, and c-met cell markers, but do not express the CD45 or c-Kit cellmarkers.

It is another object of the present invention to provide methods forisolating and enriching muscle-derived progenitor cells from a startingmuscle cell population. These methods result in the enrichment of MDCsthat have long-term survivability after transplantation or introductioninto a site of soft tissue. The MDC population according to the presentinvention is particularly enriched with cells that express progenitorcell markers, such as desmin, M-cadherin, MyoD, myogenin, CD34, andBcl-2. This MDC population also expresses the Flk-1, Sca-1, MNF, andc-met cell markers, but does not express the CD45 or c-Kit cell markers.

It is yet another object of the present invention to provide methods ofusing MDCs and compositions comprising MDCs for the augmentation ofmuscle soft tissue, or non-muscle soft tissue, including smooth muscle,and various organ tissues, without the need for polymer carriers orspecial culture media for transplantation. Such methods include theadministration of MDC compositions by introduction into soft tissue, forexample by direct injection into tissue, or by systemic distribution ofthe compositions. Preferably, soft tissue includes non-bone bodytissues. More preferably, soft tissue includes non-striated muscle andnon-bone body tissues. Most preferably, soft tissue includes non-muscle,non-bone body tissues. As used herein, augmentation refers to filling,bulking, supporting, enlarging, extending, or increasing the size ormass of body tissue.

It is another object of the present invention to provide methods ofaugmenting soft tissue, either muscle-derived soft tissue, ornon-muscle-derived soft tissue, following injury, wounding, surgeries,traumas, non-traumas, or other procedures that result in fissures,openings, depressions, wounds, and the like, in the skin or in internalsoft tissues or organs.

It is yet another object of the present invention to provide MDC-basedtreatments for gastroesophageal reflux symptoms and conditions.Pharmaceutical compositions comprising MDCs and compositions comprisingMDCs may be used for the treatment of gastro-esophageal pathologies.These pharmaceutical compositions comprise isolated MDCs. These MDCs maybe subsequently expanded by cell culture after isolation. In oneembodiment of the invention, these MDCs are frozen prior to delivery toa subject in need of the pharmaceutical composition.

In one embodiment, when the MDCs and compositions thereof are used totreat gastroesophageal reflux they are injected directly into theesophagus. Preferably, they may be injected into the lower esophagealsphincter. In another embodiment, MDCs and compositions thereof are usedto improve at least one symptom of gastro-esophageal reflux disease.These symptoms include heart burn, asthma, acid reflux, persistent sorethroat, hoarseness, chronic cough, chest pain, and feeling like there isa lump in the throat.

MDCs are isolated from a biopsy of skeletal muscle. In one embodiment,the skeletal muscle from the biopsy may be stored for 1-6 days. In oneaspect of this embodiment, the skeletal muscle from the biopsy is storedat 4° C. The MDCs are then isolated using the pre-plate or the singleplate technique.

Using the pre-plate technique, a suspension of skeletal muscle cellsfrom skeletal muscle tissue is plated in a first container to whichfibroblast cells of the skeletal muscle cell suspension adhere.Non-adherent cells are then re-plated in a second container, wherein thestep of re-plating is after about 15 to about 20% of cells have adheredto the first container. This replating step must be repeated at leastonce. The MDCs are thereby isolated and may be administered to theesophagus of the mammalian subject.

Using the single plate technique, the cells are minced, and digestedusing a collagenase, dispase, another enzyme or a combination ofenzymes. After washing the enzyme from the cells, the cells are culturedin a flask in culture medium for between about 30 and about 120 minutes.During this period of time, the “rapidly adhering cells” stick to thewalls of the flask or container, while the “slowly adhering cells” orMDCs remain in suspension. The “slowly adhering cells” are transferredto a second flask or container and cultured therein for a period of 1-3days. During this second period of time the “slowly adhering cells” orMDCs stick to the walls of the second flask or container.

In another embodiment of the invention, these MDCs are expanded to anynumber of cells. In a preferred aspect of this embodiment, the cells areexpanded in new culture media for between about 10 and 20 days. Morepreferably, the cells are expanded for 17 days.

The MDCs, whether expanded or not expanded, may be preserved in order tobe transported or stored for a period of time before use. In oneembodiment, the MDCs are frozen. Preferably, the MDCs are frozen atbetween about −20 and −90° C. More preferably, the MDCs are frozen atabout −80° C. These frozen MDCs are used as a pharmaceuticalcomposition.

It is yet another object of the present invention to provide MDC-basedtreatments for increase of lower esophageal sphincter pressure that thelower esophageal sphincter can exert. Pharmaceutical compositionscomprising MDCs and compositions comprising MDCs may be used for theincrease of lower esophageal sphincter pressure. These pharmaceuticalcompositions comprise isolated MDCs. These MDCs may be subsequentlyexpanded by cell culture after isolation. In one embodiment of theinvention, these MDCs are frozen prior to delivery to a subject in needof the pharmaceutical composition.

In one embodiment, when the MDCs and compositions thereof are used toincrease the lower esophageal sphincter pressure in a mammalian subject,they are injected directly into the esophagus. Preferably, they may beinjected into the lower esophageal sphincter. In another embodiment,MDCs and compositions thereof are used to increase the lower esophagealsphincter pressure in a mammalian subject at least 10%, or morepreferably 20%, or more preferably 30% or more preferably 40% or morepreferably 50% or more preferably 60%, or more preferably 70%, or morepreferably 80% or more preferably 90% or more preferably 100% or morepreferably 110%.

MDCs are isolated from a biopsy of skeletal muscle. In one embodiment,the skeletal muscle from the biopsy may be stored for 1-6 days. In oneaspect of this embodiment, the skeletal muscle from the biopsy is storedat 4° C. The MDCs are then isolated using the pre-plate or the singleplate technique.

Using the pre-plate technique, a suspension of skeletal muscle cellsfrom skeletal muscle tissue is plated in a first container to whichfibroblast cells of the skeletal muscle cell suspension adhere.Non-adherent cells are then re-plated in a second container, wherein thestep of re-plating is after about 15 to about 20% of cells have adheredto the first container. This replating step must be repeated at leastonce. The MDCs are thereby isolated and may be administered to theesophagus of the mammalian subject.

Using the single plate technique, the cells are minced, and digestedusing a collagenase, dispase, another enzyme or a combination ofenzymes. After washing the enzyme from the cells, the cells are culturedin a flask in culture medium for between about 30 and about 120 minutes.During this period of time, the “rapidly adhering cells” stick to thewalls of the flask or container, while the “slowly adhering cells” orMDCs remain in suspension. The “slowly adhering cells” are transferredto a second flask or container and cultured therein for a period of 1-3days. During this second period of time the “slowly adhering cells” orMDCs stick to the walls of the second flask or container.

In another embodiment of the invention, these MDCs are expanded to anynumber of cells. In a preferred aspect of this embodiment, the cells areexpanded in new culture media for between about 10 and 20 days. Morepreferably, the cells are expanded for 17 days.

The MDCs, whether expanded or not expanded, may be preserved in order tobe transported or stored for a period of time before use. In oneembodiment, the MDCs are frozen. Preferably, the MDCs are frozen atbetween about −20 and −90° C. More preferably, the MDCs are frozen atabout −80° C. These frozen MDCs are used as a pharmaceuticalcomposition.

Additional objects and advantages afforded by the present invention willbe apparent from the detailed description and exemplificationhereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

The appended drawings of the figures are presented to further describethe invention and to assist in its understanding through clarificationof its various aspects.

FIGS. 1A and 1B illustrate the results of lower esophageal (FIG. 1A) andanal sphincter (FIG. 1B) soft tissue augmentation utilizing injectionsof MDCs compositions. Injections were made into the gastroesophagealjunction or anal sphincter. At day 3 post-injection, tissue samples wereobtained and prepared for analysis. MDCs are indicated byβ-galactosidase staining. FIG. 1A shows injected tissues at 100×magnification; FIG. 1B shows injected tissues at 40× magnification.FIGS. 1A and 1B demonstrates that MDC injections maintained the loweresophageal sphincter and anal sphincter soft tissue augmentation for upto 3 days following injection.

FIGS. 2A-2C show confocal fluorescent micrographs of rat pylorus onemonth after injection with DiI labeled rat MDCs. FIG. 2A shows a DiIlabeled micrograph. FIG. 2B shows a DiI labeled, a smooth muscle actinlabeled and a merged micrograph from left to right. FIG. 2C shows a DiIlabeled, skeletal muscle myosin labeled and a merged micrograph fromleft to right.

FIG. 3 is a graph showing dose-response effect of acetylcholine-inducedcontractions of rat pyloric circular muscle strips 4 weeks after MDCtransplantation or vehicle injection (SHAM).

FIG. 4A is a bar graph that shows lower esophageal sphincter (LES)pressure measured in Beagle dogs before and 4 weeks aftertransplantation of dog MDCs.

FIG. 4B is a bar graph showing dog LES pH monitoring in one Beagle dogmeasured before (clear column) and 2 weeks after MDC transplantation(filled columns).

FIG. 5A-C show confocal fluorescent micrographs of representative imagesof grafted dog MDC in cross sections of dog LES 4 weeks aftertransplantation. DiI-labeled MDC are shown in red. Immunofluorescenceanalysis for the expression of skeletal muscle myosin (B) and smoothmuscle actin (C). Magnification=20× (A) and 40× (B and C). LM:Longitudinal Muscle; CM: Circular Muscle; MM: Muscularis Mucosa.

DETAILED DESCRIPTION OF THE INVENTION Muscle-Derived Cells andCompositions

The present invention provides MDCs comprised of early progenitor cells(also termed muscle-derived progenitor cells or muscle-derived stemcells herein) that show long-term survival rates followingtransplantation into body tissues, preferably soft tissues. To obtainthe MDCs of this invention, a muscle explant, preferably skeletalmuscle, is obtained from an animal donor, preferably from a mammal,including rats, dogs and humans. This explant serves as a structural andfunctional syncytium including “rests” of muscle precursor cells (T. A.Partridge et al., 1978, Nature 73:306 8; B. H. Lipton et al., 1979,Science 205:12924).

Cells isolated from primary muscle tissue contain mixture offibroblasts, myoblasts, adipocytes, hematopoietic, and muscle-derivedprogenitor cells. The progenitor cells of a muscle-derived populationcan be enriched using differential adherence characteristics of primarymuscle cells on collagen coated tissue flasks, such as described in U.S.Pat. No. 6,866,842 of Chancellor et al. Cells that are slow to adheretend to be morphologically round, express high levels of desmin, andhave the ability to fuse and differentiate into multinucleated myotubesU.S. Pat. No. 6,866,842 of Chancellor et al.). A subpopulation of thesecells was shown to respond to recombinant human bone morphogenic protein2 (rhBMP-2) in vitro by expressing increased levels of alkalinephosphatase, parathyroid hormone dependent 3′,5′-cAMP, and osteogeniclineage and myogenic lineages (U.S. Pat. No. 6,866,842 of Chancellor etal.; T. Katagiri et al., 1994, J. Cell Biol., 127:1755 1766).

In one embodiment of the invention, a preplating procedure may be usedto differentiate rapidly adhering cells from slowly adhering cells(MDCs). In accordance with the present invention, populations of rapidlyadhering cells (PP1-4) and slowly adhering, round MDCs (PP6) wereisolated and enriched from skeletal muscle explants and tested for theexpression of various markers using immunohistochemistry to determinethe presence of pluripotent cells among the slowly adhering cells(Example 1; patent application U.S. Ser. No. 09/302,896 of Chancellor etal.). The PP6 cells expressed myogenic markers, including desmin, MyoD,and Myogenin. The PP6 cells also expressed c-met and MNF, two geneswhich are expressed at an early stage of myogenesis (J. B. Miller etal., 1999, Curr. Top. Dev. Biol. 43:191 219). The PP6 showed a lowerpercentage of cells expressing M-cadherin, a satellite cell-specificmarker (A. Irintchev et al., 1994, Development Dynamics 199:326 337),but a higher percentage of cells expressing Bcl-2, a marker limited tocells in the early stages of myogenesis (J. A. Dominov et al., 1998, J.Cell Biol. 142:537 544). The PP6 cells also expressed CD34, a markeridentified with human hematopoietic progenitor cells, as well as stromalcell precursors in bone marrow (R. G. Andrews et al., 1986, Blood 67:842845; C. I. Civin et al., 1984, J. Immunol. 133:157 165; L. Fina et al,1990, Blood 75:2417 2426; P. J. Simmons et al., 1991, Blood 78:28482853). The PP6 cells also expressed Flk-1, a mouse homologue of humanKDR gene which was recently identified as a marker of hematopoieticcells with stem cell-like characteristics (B. L. Ziegler et al., 1999,Science 285:1553 1558). Similarly, the PP6 cells expressed Sca-1, amarker present in hematopoietic cells with stem cell-likecharacteristics (M. van de Rijn et al., 1989, Proc. Natl. Acad. Sci. USA86:4634 8; M. Osawa et al., 1996, J. Immunol. 156:3207 14). However, thePP6 cells did not express the CD45 or c-Kit hematopoietic stem cellmarkers (reviewed in L K. Ashman, 1999, Int. J. Biochem. Cell. Biol.31:1037 51; G. A. Koretzky, 1993, FASEB J. 7:420 426).

One embodiment of the present invention is the PP6 population ofmuscle-derived progenitor cells having the characteristics describedherein. These muscle-derived progenitor cells express the desmin, CD34,and Bcl-2 cell markers. In accordance with the present invention, thePP6 cells are isolated by the techniques described herein (Example 1) toobtain a population of muscle-derived progenitor cells that havelong-term survivability following transplantation. The PP6muscle-derived progenitor cell population comprises a significantpercentage of cells that express progenitor cell markers such as desmin,CD34, and Bcl-2. In addition, PP6 cells express the Flk-1 and Sca-1 cellmarkers, but do not express the CD45 or c-Kit markers. Preferably,greater than 95% of the PP6 cells express the desmin, Sca-1, and Flk-1markers, but do not express the CD45 or c-Kit markers. It is preferredthat the PP6 cells are utilized within about 1 day or about 24 hoursafter the last plating.

In a preferred embodiment, the rapidly adhering cells and slowlyadhering cells (MDCs) are separated from each other using a singleplating technique. One such technique is described in Example 2. First,cells are provided from a skeletal muscle biopsy. The biopsy need onlycontain about 100 mg of cells. Biopsies ranging in size from about 50 mgto about 500 mg are used in certain embodiments according to both thepre-plating and single plating methods of the invention. Further,biopsies of 50, 100, 110, 120, 130, 140, 150, 200, 250, 300, 400 and 500mg may be used according to both the pre-plating and single platingmethods of the invention.

In a preferred embodiment of the invention, the tissue from the biopsyis then stored for 1 to 7 days. This storage is at a temperature fromabout room temperature to about 4° C. This waiting period causes thebiopsied skeletal muscle tissue to undergo stress. While this stress isnot necessary for the isolation of MDCs using this single platetechnique, it seems that using the wait period results in a greateryield of MDCs.

Tissue from the biopsies is minced and centrifuged. The pellet isresuspended and digested using a digestion enzyme. Enzymes that may beused include collagenase, dispase or combinations of these enzymes.After digestion, the enzyme is washed off of the cells. The cells aretransferred to a flask in culture media for the isolation of the rapidlyadhering cells. Many culture media may be used. Particularly preferredculture media include those that are designed for culture of endothelialcells including Cambrex Endothelial Growth Medium. This medium may besupplemented with other components including fetal bovine serum, IGF-1,bFGF, VEGF, EGF, hydrocortisone, heparin, and/or ascorbic acid. Othermedia that may be used in the single plating technique include InCellM310F medium. This medium may be supplemented as described above, orused unsupplemented.

The step for isolation of the rapidly adhering cells may require culturein flask for a period of time from about 30 to about 120 minutes. Therapidly adhering cells adhere to the flask in 30, 40, 50, 60, 70, 80,90, 100, 110 or 120 minutes. After they adhere, the slowly adheringcells are separated from the rapidly adhering cells from removing theculture media from the flask to which the rapidly adhering cells areattached to.

The culture medium removed from this flask is then transferred to asecond flask. The cells may be centrifuged and resuspended in culturemedium before being transferred to the second flask. The cells arecultured in this second flask for between 1 and 3 days. Preferably, thecells are cultured for two days. During this period of time, the slowlyadhering cells (MDCs) adhere to the flask. After the MDCs have adhered,the culture media is removed and new culture media is added so that theMDCs can be expanded in number. The MDCs may be expanded in number byculturing them for from about 10 to about 20 days. The MDCs may beexpanded in number by culturing them for 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20 days. Preferably, the MDCs are subject to expansion culturefor 17 days.

As an alternative to the pre-plating and single plating methods, theMDCs of the present invention can be isolated by fluorescence-activatedcell sorting (FACS) analysis using labeled antibodies against one ormore of the cell surface markers expressed by the MDCs (C. Webster etal., 1988, Exp. Cell. Res. 174:252 65; J. R. Blanton et al., 1999,Muscle Nerve 22:43 50). For example, FACS separation can be performedusing labeled antibodies to directed to CD34, Flk-1, Sca-1, and/or theother cell-surface markers described herein to select a population ofPP6-like cells that exhibit long-term survivability when introduced intothe host tissue. Also encompassed by the present invention is the use ofone or more fluorescence-detection labels, for example, fluorescein orrhodamine, for antibody detection of different cell marker proteins.

Using any of the MDCs isolation methods described above, MDCs that areto be transported, or are not going to be used for a period of time maybe preserved using methods known in the art. More specifically, theisolated MDCs may be frozen to a temperature ranging from about −25 toabout −90° C. Preferably, the MDCs are frozen at about −80° C., on dryice for delayed use or transport. The freezing may be done with anycryopreservation medium known in the art.

Muscle-Derived Cell-Based Treatments

In one embodiment of the present invention, the MDCs are isolated from askeletal muscle source and introduced or transplanted into a muscle ornon-muscle soft tissue site of interest. Advantageously, the MDCs of thepresent invention are isolated and enriched to contain a large number ofprogenitor cells showing long-term survival following transplantation.In addition, the muscle-derived progenitor cells of this inventionexpress a number of characteristic cell markers, such desmin, CD34, andBcl-2. Furthermore, the muscle-derived progenitor cells of thisinvention express the Sca-1, and Flk-1 cell markers, but do not expressthe CD45 or c-Kit cell markers (see Example 1).

MDCs and compositions comprising MDCs of the present invention can beused to repair, treat, or ameliorate various aesthetic or functionalconditions (e.g. defects) through the augmentation of muscle ornon-muscle soft tissues. In particular, such compositions can be used assoft-tissue bulking agents for the treatment of gastroesophageal refluxsymptoms or conditions.

For MDC-based treatments, a skeletal muscle explant is preferablyobtained from an autologous or heterologous human or animal source. Anautologous animal or human source is more preferred. MDC compositionsare then prepared and isolated as described herein. To introduce ortransplant the MDCs and/or compositions comprising the MDCs according tothe present invention into a human or animal recipient, a suspension ofmononucleated muscle cells is prepared. Such suspensions containconcentrations of the muscle-derived progenitor cells of the inventionin a physiologically-acceptable carrier, excipient, or diluent. Forexample, suspensions of MDCs for administering to a subject can comprise10⁸ to 10⁹ cells/ml in a sterile solution of complete medium modified tocontain the subject's serum, as an alternative to fetal bovine serum.Alternatively, MDC suspensions can be in serum-free, sterile solutions,such as cryopreservation solutions (Celox Laboratories, St. Paul, Minn.)The MDC suspensions can then be introduced e.g., via injection, into oneor more sites of the donor tissue.

The described cells can be administered as a pharmaceutically orphysiologically acceptable preparation or composition containing aphysiologically acceptable carrier, excipient, or diluent, andadministered to the tissues of the recipient organism of interest,including humans and non-human animals. The MDC-containing compositioncan be prepared by resuspending the cells in a suitable liquid orsolution such as sterile physiological saline or other physiologicallyacceptable injectable aqueous liquids. The amounts of the components tobe used in such compositions can be routinely determined by those havingskill in the art.

The MDCs or compositions thereof can be administered by placement of theMDC suspensions onto absorbent or adherent material, i.e., a collagensponge matrix, and insertion of the MDC-containing material into or ontothe site of interest. Alternatively, the MDCs can be administered byparenteral routes of injection, including subcutaneous, intravenous,intramuscular, and intrasternal. Other modes of administration include,but are not limited to, intranasal, intrathecal, intracutaneous,percutaneous, enteral, and sublingual. In one embodiment of the presentinvention, administration of the MDCs can be mediated by endoscopicsurgery.

For injectable administration, the composition is in sterile solution orsuspension or can be resuspended in pharmaceutically- andphysiologically-acceptable aqueous or oleaginous vehicles, which maycontain preservatives, stabilizers, and material for rendering thesolution or suspension isotonic with body fluids (i.e. blood) of therecipient. Non-limiting examples of excipients suitable for use includewater, phosphate buffered saline, pH 7.4, 0.15 M aqueous sodium chloridesolution, dextrose, glycerol, dilute ethanol, and the like, and mixturesthereof. Illustrative stabilizers are polyethylene glycol, proteins,saccharides, amino acids, inorganic acids, and organic acids, which maybe used either on their own or as admixtures. The amounts or quantities,as well as the routes of administration used, are determined on anindividual basis, and correspond to the amounts used in similar types ofapplications or indications known to those of skill in the art.

To optimize transplant success, the closest possible immunological matchbetween donor and recipient is desired. If an autologous source is notavailable, donor and recipient Class I and Class II histocompatibilityantigens can be analyzed to determine the closest match available. Thisminimizes or eliminates immune rejection and reduces the need forimmunosuppressive or immunomodulatory therapy. If required,immunosuppressive or immunomodulatory therapy can be started before,during, and/or after the transplant procedure. For example, cyclosporinA or other immunosuppressive drugs can be administered to the transplantrecipient. Immunological tolerance may also be induced prior totransplantation by alternative methods known in the art (D. J. Watt etal., 1984, Clin. Exp. Immunol. 55:419; D. Faustman et al., 1991, Science252:1701).

Consistent with the present invention, the MDCs are administered to thedigestive system (i.e., mouth, tongue, esophagus, stomach, liver,pancreas, gall bladder, intestine, anus, etc.).

Conditions of the lumen: In another embodiment, the MDCs andcompositions thereof according to the present invention have furtherutility as treatments for conditions of the lumen in an animal or mammalsubject, including humans. Specifically, the muscle-derived progenitorcells are used for completely or partially blocking, enhancing,enlarging, sealing, repairing, bulking, or filling various biologicallumens or voids within the body. Lumens include, without limitation,intestine, stomach and esophagus. Voids may include, without limitation,various tissue wounds (i.e., loss of muscle and soft tissue bulk due totrauma; destruction of soft tissue due to penetrating projectiles suchas a stab wound or bullet wound; loss of soft tissue from disease ortissue death due to surgical removal of the tissue), lesions, fissures,diverticulae, cysts, fistulae, and other undesirable or unwanteddepressions or openings that may exist within the body of an animal ormammal, including humans. For the treatment of conditions of the lumen,the MDCs are prepared as disclosed herein and then administered, e.g.via injection or intravenous delivery, to the lumenal tissue to fill orrepair the void. The number of MDCs introduced is modulated to repairlarge or small voids in a soft tissue environment, as required.

Conditions of the sphincter: The MDCs and compositions thereof accordingto the present invention can also be used for the treatment of asphincter injury, weakness, disease, or dysfunction in an animal ormammal, including humans. In particular, the MDCs are used to augmenttissues of the esophageal, anal and pyloric sphincter. Preferably, thesphincter is the lower esophageal sphincter. More specifically, thepresent invention provides soft tissue augmentation treatments forgastroesophageal reflux symptoms. For the treatment of sphincterdefects, the MDCs are prepared as described herein and then administeredto the sphincter tissue, e.g. via injection, to provide additional bulk,filler, or support. The number of MDCs introduced is modulated toprovide varying amounts of bulking material as required. For example,about 1 to about 5×10⁶ MDCs are used to provide augmentation for anapproximately 5 mm region of the gastroesophageal junction or anapproximately 5-10 mm region of the anal sphincter (see Example 3). Thecells can be engrafted so as to reside within the muscle wall and/ormuscularis mucosa of the treated sphincter region. As well, in thetreatment of a lower esophageal sphincter, the cellular grafting can beeffective to increase the pressure of the sphincter, and/or to reduceacid reflux into the esophagus, e.g. as measured by the fraction of timethat the affected lower esophagus exhibits a pH of less than about 4.

Muscle augmentation and contractility: In yet another embodiment of thepresent invention, the MDCs and compositions thereof are used for thetreatment of muscle conditions in a human or animal subject. Inparticular, the MDCs can be used to augment smooth muscles to treatweakness or dysfunction caused by injury, disease, inactivity, oranoxia- or surgery-induced trauma.

For muscle augmentation or treatment of muscle-related conditions, theMDCs are prepared as described above and are administered, e.g. viainjection, into muscle tissue to provide additional bulk, filler, orsupport. As is appreciated by the skilled practitioner, the number ofMDCs introduced is modulated to provide varying amounts of bulkingmaterial, as needed or required.

In addition, the MDCs and compositions thereof can be used to affectcontractility in smooth muscle tissue, such as gastrointestinal tissue,and esophageal tissue, as example. The present invention also embracesthe use of MDCs of the invention in restoring muscle contraction, and/orameliorating or overcoming smooth muscle contractility problems, suchdecreased gastrointestinal motility, including the esophagus, stomachand intestine smooth muscle. A specific, yet nonlimiting example of acondition that the MDCs of the invention can improve, reduce, or correctis gastroparesis, i.e., poor motility and emptying of the stomach.

EXAMPLES Example 1. MDC Enrichment, Isolation and Analysis According tothe Pre-Plating Method

Enrichment and isolation of MDCs: MDCs were prepared as described (U.S.Pat. No. 6,866,842 of Chancellor et al.). Muscle explants were obtainedfrom the hind limbs of a number of sources, namely from 3-week-old mdx(dystrophic) mice (C57BL/10ScSn mdx/mdx, Jackson Laboratories), 4 6week-old normal female SD (Sprague Dawley) rats, or SCID (severecombined immunodeficiency) mice. The muscle tissue from each of theanimal sources was dissected to remove any bones and minced into aslurry. The slurry was then digested by 1 hour serial incubations with0.2% type XI collagenase, dispase (grade II, 240 unit), and 0.1% trypsinat 37° C. The resulting cell suspension was passed through 18, 20, and22 gauge needles and centrifuged at 3000 rpm for 5 minutes.Subsequently, cells were suspended in growth medium (DMEM supplementedwith 10% fetal bovine serum, 10% horse serum, 0.5% chick embryo extract,and 2% penicillin/streptomycin). Cells were then preplated incollagen-coated flasks (U.S. Pat. No. 6,866,842 of Chancellor et al.).After approximately 1 hour, the supernatant was removed from the flaskand re-plated into a fresh collagen-coated flask. The cells whichadhered rapidly within this 1 hour incubation were mostly fibroblasts(Z. Qu et al., supra; U.S. Pat. No. 6,866,842 of Chancellor et al.). Thesupernatant was removed and re-plated after 30-40% of the cells hadadhered to each flask. After approximately 5-6 serial platings, theculture was enriched with small, round cells, designated as PP6 cells,which were isolated from the starting cell population and used infurther studies. The adherent cells isolated in the early platings werepooled together and designated as PP1-4 cells.

The mdx PP1-4, mdx PP6, normal PP6, and fibroblast cell populations wereexamined by immunohistochemical analysis for the expression of cellmarkers. The results of this analysis are shown in Table 1.

TABLE 1 Cell markers expressed in PP1-4 and PP6 cell populations. mdxPP1-4 mdx PP6 nor PP6 cells cells cells fibroblasts desmin +/− + + −CD34 − + + − Bcl-2 (−) + + − Flk-1 na + + − Sca-1 na + + − M-cadherin−/+ −/+ −/+ − MyoD −/+ +/− +/− − myogenin −/+ +/− +/− − Mdx PP1-4, mdxPP6, normal PP6, and fibroblast cells were derived by preplatingtechnique and examined by immunohistochemical analysis. “−” indicatesless than 2% of the cells showed expression; “(−)”; “−/+” indicates5-50% of the cells showed expression; “+/−” indicates ~40-80% of thecells showed expession; “+” indicates that >95% of the cells showedexpression; “nor” indicates normal cells; “na” indicates that theimmunohistochemical data is not available.

It is noted that both mdx and normal mice showed identical distributionof all of the cell markers tested in this assay. Thus, the presence ofthe mdx mutation does not affect the cell marker expression of theisolated PP6 muscle-cell derived population.

MDCs were grown in proliferation medium containing DMEM (Dulbecco'sModified Eagle Medium) with 10% FBS (fetal bovine serum), 10% HS (horseserum), 0.5% chick embryo extract, and 1% penicillin/streptomycin, orfusion medium containing DMEM supplemented with 2% fetal bovine serumand 1% antibiotic solution. All media supplies were purchased throughGibco Laboratories (Grand Island, N.Y.).

Example 2. MDC Enrichment, Isolation and Analysis According to theSingle Plate Method

Populations of rapidly- and slowly-adhering MDCs were isolated fromskeletal muscle of a mammalian subject. The subject may be a human, rat,dog or other mammal. Biopsy size ranged from 42 to 247 mg.

Skeletal muscle biopsy tissue is immediately placed in cold hypothermicmedium (HypoThermosol (BioLife) supplemented with gentamicin sulfate(100 ng/ml, Roche)) and stored at 4° C. After 3 to 7 days, biopsy tissueis removed from storage and production is initiated. Any connective ornon-muscle tissue is dissected from the biopsy sample. The remainingmuscle tissue that is used for isolation is weighed. The tissue isminced in Hank's Balanced Salt Solution (HBSS), transferred to a conicaltube, and centrifuged (2,500×g, 5 minutes). The pellet is thenresuspended in a Digestion Enzyme solution (Liberase Blendzyme 4(0.4-1.0 U/mL, Roche)). 2 mL of Digestion Enzyme solution is used per100 mg of biopsy tissue and is incubated for 30 minutes at 37° C. on arotating plate. The sample is then centrifuged (2,500×g, 5 minutes). Thepellet is resuspended in culture medium and passed through a 70 μm cellstrainer. The culture media used for the procedures described in thisExample was Cambrex

Endothelial Growth Medium EGM-2 basal medium supplemented with thefollowing components: i. 10% (v/v) fetal bovine serum, and ii. CambrexEGM-2 SingleQuot Kit, which contains: Insulin Growth Factor-1 (IGF-1),Basic Fibroblast Growth Factor (bFGF), Vascular Endothelial GrowthFactor (VEGF), Epidermal Growth Factor (EGF), Hydrocortisone, Heparin,and Ascorbic Acid. The filtered cell solution is then transferred to aT25 culture flask and incubated for 30-120 minutes at 37° C. in 5% CO₂.Cells that attach to this flask are the “rapidly-adhering cells”.

After incubation, the cell culture supernatant is removed from the T25flask and placed into a 15 mL conical tube. The T25 culture flask isrinsed with 2 mL of warmed culture medium and transferred to theaforementioned 15 mL conical tube. The 15 mL conical tube is centrifuged(2,500×g, 5 minutes). The pellet is resuspended in culture medium andtransferred to a new T25 culture flask. The flask is incubated for daysat 37° C. in 5% CO₂ (cells that attach to this flask are the“slowly-adhering cells”). After incubation, the cell culture supernatantis aspirated and new culture medium is added to the flask. The flask isthen returned to the incubator for expansion. Standard culture passagingis carried out from here on to maintain the cell confluency in theculture flask at less than 50%. Trypsin-EDTA (0.25%, Invitrogen) is usedto detach the adherent cells from the flask during passage. Typicalexpansion of the “slowly-adhering cells” takes an average of 17 days(starting from the day production is initiated) to achieve an averagetotal viable cell number of 37 million cells.

Once the desired cell number is achieved, the cells are harvested fromthe flask using Trypsin-EDTA and centrifuged (2,500×g, 5 minutes). Thepellet is resuspended in BSS-P solution (HBSS supplemented with humanserum albumin (2% v/v, Sera Care Life)) and counted. The cell solutionis then centrifuged again (2,500×g, 5 minutes), resuspended withCryopreservation Medium (CryoStor (Biolife) supplemented with humanserum albumin (2% v/v, Sera Care Life Sciences)) to the desired cellconcentration, and packaged in the appropriate vial for cryogenicstorage. The cryovial is placed into a freezing container and placed inthe −80° C. freezer. Cells are administered by thawing the frozen cellsuspension at room temperature with an equal volume of physiologicsaline and injected directly (without additional manipulation). Thelineage characterization of the slowly adhering cell populations shows:Myogenic (87.4% CD56+, 89.2% desmin+), Endothelial (0.0% CD31+),Hematopoietic (0.3% CD45+), and Fibroblast (6.8% CD90+/CD56-).

Following disassociation of the skeletal muscle biopsy tissue, twofractions of cells were collected based on their rapid or slow adhesionto the culture flasks. The cells were then expanded in culture withgrowth medium and then frozen in cryopreservation medium (3×10⁵ cells in15 μl) in a 1.5 ml eppendorf tube. For the control group, 15 μl ofcryopreservation medium alone was placed into the tube. These tubes werestored at −80° C. until injection. Immediately prior to injection, atube was removed from storage, thawed at room temperature, andresuspended with 15 μl of 0.9% sodium chloride solution. The resulting30 μl solution was then drawn into a 0.5 cc insulin syringe with a 30gauge needle. The investigator performing the surgery and injection wasblinded to the contents of the tubes.

Cell count and viability was measured using a Guava flow cytometer andViacount assay kit (Guava). CD56 was measured by flow cytometry (Guava)using PE-conjugated anti-CD56 antibody (1:50, BD Pharmingen) andPE-conjugated isotype control monoclonal antibody (1:50, BD Pharmingen).Desmin was measured by flow cytometry (Guava) on paraformaldehyde-fixedcells (BD Pharmingen) using a monoclonal desmin antibody (1:100, Dako)and an isotype control monoclonal antibody (1:200, BD Pharmingen).Fluorescent labeling was performed using a Cy3-conjugated anti-mouse IgGantibody (1:250, Sigma). In between steps, the cells were washed withpermeabilization buffer (BD Pharmingen). For creatine kinase (CK) assay,1×10⁵ cells were plated per well into a 12 well plate indifferentiation-inducing medium. Four to 6 days later, the cells wereharvested by trypsinization and centrifuged into a pellet. The celllysis supernatant was assayed for CK activity using the CK Liqui-UV kit(Stanbio).

Example 3. Soft Tissue Augmentation of the Gastro-Esophageal Junctionand Anal Sphincter

Sprague-Dawley (SD) rats were prepared for surgery by anesthetizing withhalothane using standard methods, and washing the surgical site withBetadine® solution. A midline abdomen incision was made to expose thegastroesophageal junction and anal sphincter. The soft tissue wasinjected with 10 μl of a suspension of muscle-derived progenitor cells,prepared pursuant to the methods of Example 1, in HBSS (1-1.5×10⁶ cells)using a Hamilton microsyringe. At day 3 post-injection, the areasurrounding each injection site was excised, prepared for histochemicalanalysis, stained for β-galactosidase to determine the location andviability of the cells carrying the LacZ marker, examinedmicroscopically, and photographed. Results of these experimentsdemonstrate that MDC compositions can be used as esophageal and analsphincter bulking materials (FIGS. 1A and 1B) for the treatment ofgastroesophageal reflux or fecal incontinence symptoms or conditions.

Example 4. MDC Implantation in the Lower Esophageal Sphincter for theTreatment of Gastro-Esophageal Reflux Disorder (GERD)

Most patients with GERD who are being considered for surgery have lowlower esophageal sphincter (LES) pressure. We hypothesized thatauto-transplantation of skeletal muscle-derived cells (MDC) into the LESmay offer the ideal bulking therapy. To test this, we have performedexperiments to test the potential of MDCs to survive and differentiatewithin the gastro intestinal smooth muscle in order to gain furtherknowledge on the biology of skeletal muscle transplantation in GI smoothmuscle sphincters as well as to test the safety and feasibility ofendoscopic injection of MDC in a large animal model.

Adult male Sprague-Dawley (SD) rats and adult male Beagle dogs wereused. Rat-derived and dog-derived MDC were isolated using a single platetechnique similar to the technique described in Example 2. MDCs werelabeled with DiI, a lipophilic membrane stain that diffuses across thewhole cell, (Invitrogen/Molecular Probes) before transplantation inorder to be able to visualize the cells in the host tissue.Differentiation of grafted cells was assessed by immunofluorescenceusing specific antibodies to markers of the smooth muscle phenotype(smooth muscle actin) and of the skeletal muscle phenotype (skeletalmuscle myosin).

Rat Experiments.

DiI-labeled rat-derived MDCs were injected bilaterally in the pyloricwall of rats using a 10 μl Hamilton syringe and survival anddifferentiation was assessed 1 month post-transplantation. Grafted cellswere visualized based on DiI fluorescence and were found to be localizedwithin the muscle wall and in the muscularis mucosa, as shown in FIG.2A. Immunofluorescence analysis revealed weak expression of skeletalmuscle myosin in grafted MDC and no expression of smooth muscle actin,as shown in FIGS. 2B (smooth muscle actin) and 2C (skeletal musclemyosin). MDC can survive and integrate into GI smooth muscle and theyhave potential for the treatment of a variety of conditions includingGERD.

MDC Transplantation in a Canine Model of GERD.

In the first of a series of ongoing experiments, 4.0×10⁶ of labeledcanine MDC were injected into the LES of a Beagle dog using a standardvariceal sclerotherapy needle delivered through an endoscope. The dogwas treated with daily cyclosporine and two weeks later pH monitoringrepeated and the esophagus examined histologically. A significantreduction of acid reflux was observed with the fraction of time withpH<4 decreasing from 26.5% to 1.5%. Transplanted MDC were seen (byimmunofluorescence staining) adding bulk to the lower esophageal area,and were well integrated into the surrounding tissue particularly in themuscularis mucosa.

Example 5. MDC Implantation in the Lower Esophageal Sphincter IncreaseContractility

Organ bath analysis of the rat pyloric muscle revealed a significantincrease in muscle contraction in response to acetylcholine after MDCtransplantation as compared to saline-injected controls (FIG. 2).

Organ Bath Analysis of Pyloric Function.

Four weeks post-transplantation, rats were anesthetized and the pyloricregion dissected out. Circular muscle strips were mounted between twoL-shaped tissue hooks in 5 mL chambers containing Krebs buffer at 37° C.and continuously bubbled with 95% O₂, 5% CO₂. (Radnoti Glass Technology,Monrovia, Calif.). Tension was monitored with an isometric forcetransducer and recorded and analyzed by a digital recording system(Biopac Systems Inc., Santa Barbara, Calif.). Strips were stretched to 2g (10 mN) and allowed to equilibrate for 30 minutes. Pyloric functionwas assessed by measuring the ability of the pyloric circular muscle tocontract in response to acetylcholine. Acetylcholine (ACh) was added tothe chambers from freshly prepared 100× stock solutions. The weight ofthe tissue was measured at the end of each session.

Comparisons between the groups was performed by measuring the area underthe curve of the ACh-induced contraction (AUCC) for 1 minute and thebaseline for 1 minute (AUCB), according to the following formula:(AUCC-AUCB)/weight of tissue (mg)=AUC/mg of tissue.

Rat-derived MDCs were labeled with CM-DiI (Invitrogen Co., Carlsbad,Calif.) according to Manufacturer's instruction to allow for theidentification of grafted cells in the host tissue. Adult maleSprague-Dawley rats were deeply anesthetized using a combination ofketamine/xylazine, a midline incision was made and the pyloric regionidentified. DiI-labeled rat MDC were suspended in Dulbecco's PBS andinjected bilaterally (50,000 cells/μl; 2 μl per site) in the pyloricwall using a 10 μl Hamilton syringe. Another set of rats receivedbilateral injections of Dulbecco's PBS and served as sham controls.

Organ bath analysis of the rat pyloric muscle revealed a significantincrease in muscle contraction in response to acetylcholine after MDCtransplantation as compared to saline-injected controls (FIG. 3).Analysis of the results by one-way ANOVA revealed a significant effectof MDC transplantation (P=0.002). Data are means+/−SEM. N=6 for MDC and7 for SHAM.

Three Beagle dogs were evaluated for the effects of MDC injection onesophageal physiology. All underwent baseline esophageal manometry tomeasure LES pressure as well as pH monitoring using a Bravo system.Subsequently, 4.0×10⁶ of DiI-labeled canine MDC were suspended in 5 mlof sterile physiological saline and injected into the LES of all threeBeagle dogs using a variceal sclerotherapy needle. The dogs were treatedwith cyclosporine daily 100 mg/day and manometry was performed 1-2 weekslater on all three dogs.

In dogs, MDC injection resulted in a significant increase in baselineLES pressure (FIG. 4A). Data are means+/−SEM; N=3. P<0.01 by Student'sT-test. Further in one dog with significant baseline acid reflux, MDCinjection resulted in a dramatic reduction of acid reflux, with thefraction of time with pH<4 decreasing from 26.5% to 1.5%; the number ofrefluxes measured was reduced from 95 in 22 hours 48 minutes to 11 in 18hours 28 minutes; the number of refluxes lasting longer than 5 minuteswas reduced from 16 in 22 hours 48 minutes to 0 in 18 hours 28 minutes;and the duration of longest reflux was reduced from 76 minutes to 2minutes. (FIG. 4B). Transplanted MDC were seen adding bulk to the loweresophageal area, and were well integrated into the surrounding tissueparticularly in the muscularis mucosa. Grafted cells showed positiveimmunoreactivity for skeletal muscle myosin and not for smooth muscleactin (FIG. 5).

All patent applications, patents, texts, and literature references citedin this specification are hereby incorporated herein by reference intheir entirety to more fully describe the state of the art to which thepresent invention pertains.

As various changes can be made in the above methods and compositionswithout departing from the scope and spirit of the invention asdescribed, it is intended that all subject matter contained in the abovedescription, shown in the accompanying drawings, or defined in theappended claims be interpreted as illustrative, and not in a limitingsense.

What is claimed is:
 1. A method of increasing lower esophageal sphincterpressure in a mammalian subject comprising: (a) isolating skeletalmuscle cells from the mammalian subject; (b) cooling the cells to atemperature lower than 10° C. and storing the cells for 1-7 days; (c)suspending the mammalian skeletal muscle cells in a first cell culturecontainer between 30 and 120 minutes; (d) decanting the media from thefirst cell culture container to a second cell culture container; (e)allowing the remaining cells in the media to attach to the walls of thesecond cell culture container; (f) isolating the cells from the walls ofthe second cell culture container, wherein the isolated cells are MDCs;(g) culturing the cells to expand their number; (h) freezing the MDCs toa temperature below −30° C.; and (i) thawing the MDCs and administeringthe MDCs to the esophagus of the mammalian subject; thereby, increasinglower esophageal sphincter pressure by at least about 50% in a mammaliansubject.
 2. The method of claim 1, wherein the skeletal muscle cells areisolated from the mammalian subject before the gastro-esophageal refluxdisease begins in the mammalian subject.
 3. The method of claim 1,wherein increase in lower esophageal sphincter pressure is increased byat least about 100%.
 4. The method of claim 1, wherein the MDCs areadministered by injecting them into the esophagus.
 5. The method ofclaim 1, wherein the MDCs are injected into the lower esophagealsphincter.
 6. The method of claim 1, wherein the mammal is a human.
 7. Amethod of increasing lower esophageal sphincter pressure in a mammaliansubject comprising: (a) isolating skeletal muscle cells from themammalian subject, (b) suspending mammalian skeletal muscle cells in afirst cell culture container for between 30 and 120 minutes; (c)decanting the media from the first cell culture container to a secondcell culture container; (d) allowing the remaining cells in the media toattach to the walls of the second cell culture container; (e) isolatingthe cells from the walls of the second cell culture container, whereinthe isolated cells are MDCs; and (f) administering the MDCs to theesophagus of the mammalian subject; thereby, increasing lower esophagealsphincter pressure in a in a mammalian subject.
 8. The method of claim7, wherein increase in lower esophageal sphincter pressure is increasedby at least about 100%.
 9. The method of claim 7, wherein the MDCs areadministered by injecting them into the esophagus.
 10. The method ofclaim 9, wherein the MDCs are injected into the lower esophagealsphincter.
 11. The method of claim 7, wherein the mammal is a human. 12.The method of claim 7, wherein the MDCs are cultured to expand theirnumber before being administered to the esophagus of the mammaliansubject.
 13. A method of increasing lower esophageal sphincter pressurein a mammalian subject comprising: (a) plating a suspension of skeletalmuscle cells from skeletal muscle tissue in a first container to whichfibroblast cells of the skeletal muscle cell suspension adhere, (b)re-plating non-adherent cells from step (a) in a second container,wherein the step of re-plating is after about 15 to about 20% of cellshave adhered to the first container; (c) repeating step (b) at leastonce; (d) isolating the skeletal muscle-derived MDCs and administeringthe MDCs to the esophagus of the mammalian subject; thereby increasinglower esophageal sphincter pressure in a mammalian subject.
 14. Themethod of claim 13, wherein increase in lower esophageal sphincterpressure is increased by at least about 100%.
 15. The method of claim13, wherein the MDCs are administered by injecting them into theesophagus.
 16. The method of claim 13, wherein the MDCs are injectedinto the lower esophageal sphincter.
 17. The method of claim 13, whereinthe mammal is a human.